This proposal focuses upon the discovery and evolutionary potential of promiscuous activities - secondary activities that occur adventitiously as a result of the highly reactive environments in active sites. Promiscuous activities normally provide no benefit to the organism. However, when conditions change, a promiscuous activity may provide the starting point for evolution of a new enzyme that has become critical for fitness. A better understanding of catalytic promiscuity will help us understand how thousands of highly effective enzymes evolved over the 3.8 billion years since the origin of life, and will provide a better foundation for protein engineering efforts, which are most effective when an enzyme with a desired activity, even if it is inefficient, is used as a starting place. This project explores the mechanisms and evolutionary potential of promiscuous activities discovered during the preceding grant period, and extends our search for prbmiscudus ehzymes using a hew strategy. Our Specific Aiifis are as follows. Aim 1. To explore the mechanistic basis of the promiscuous activities of phosphoenolpyruvate (PEP) synthase and dihydroquinate (DHQ) synthase. Aim 2. To use directed evolution to explore the evolutionary potential of the promiscuous activity of PEP synthase. Aim 3. To explore the constraints on evolution of a new enzymatic activity imposed by the need to retain the original function of the protein. Aim 4. To take a new approach to discovery of promiscuous enzymes by identifying adaptive mutations that restore viability in strains lacking genes essential for growth on glucose. Relevance to public health: New enzymes continue to evolve as humans exert novel selective pressures upon microbes by use of antibiotics in medical and agricultural settings and by release of toxic chemicals into the environment. In vitro evolution of enzymes to serve new functions is a major effort in biotechnology because enzymes are "green" catalysts, able to achieve massive rate accelerations without organic solvents, toxic heavy metal catalysts, or energy-intensive processes. Thus, the processes by which new enzyme activities evolve are of great interest for both theoretical and practical reasons.